Research On Construction Of DNA Biosensing System Based On Cationic Comb-Type Copolymers

As a genetic material of life,DNA has the function of storing and transmitting genetic information.The dynamic DNA based nanomachine has been developed through simple sequences design that utilize DNA's excellent molecular recognition properties and structural features.The output and frame strength of the machines depend on the stability of DNA hybrids,while the quick response relies on hybridization and,more specifically,strand exchange reaction rates.It is difficult to satisfy both requirements simultaneously because of the dilemma that an increase in hybrid stability generally results in a decrease in the strand exchange rates.In order to overcome these shortcomings,this paper used the cationic comb-type copolymer PLL-g-Dex as the research object to construct a DNA biosensing system,based on cationic comb-type copolymer.This paper includes three parts:In the first part,poly-(L-ysine)grafted dextran(PLL-g-Dex)copolymer was prepared by reductive amination reaction.The structure was characterized by FT-IR and 1H-NMR.The molecular weight of the PLL-g-Dex copolymer was examined by the gel permeation chromatography.The results showed that PLL-g-Dex copolymer was synthesized by reductive amination reaction successfully.The final PLL-g-Dex copolymer had a number average molecular weight of 161543 and a weight average molecular weight of 201312.The Wt%of PLL-g-Dex copolymer was 91%by 1H-NMR spectroscopy and GPC analysis.In the second part,the biosensor system of GO-DNA based on cationic comb-type copolymer was constructed by using graphene oxide as high-efficiency quencher.Through the infrared characterization of graphene oxide,it was found that graphene oxide contains many polar groups and has good hydrophilicity and can be used in the following experiments.The feasibility of the detection principle was verified by experiments.When the concentrations of GO,fully matched cDNA and PLL-g-Dex were 9?g/ml,90nM and 96nM,respectively,the specificity of CC single base mismatch in T-DNA sequence were investigated.This has some research significance in the prediction of human genetic disease.In the third part,in order to broaden the application field of PLL-g-Dex copolymer,a series of polyelectrolyte-assisted DNA mechanical nanoswitchs with rapid response were developed by native polyacrylamide gel electrophoresis,fluorescence spectroscopy and UV-melting point(Tm)measurement.To explore the dimerization process of DIS25 driven by cationic comb-type copolymer PLL-g-Dex and the dissociation kinetic process of DIS25 dimer driven by the PVS.It was found that PLL-g-Dex greatly promoted the formation of dimerization of self-complementary stem-loop DNA,and simultaneously PLL-g-Dex increased the Tm of dimer from 54.5 ? to 66.7 ?.The results proved that PLL-g-Dex had a stable effect on DIS25 dimer.The dissociation kinetics of DIS25 dimer driven by PVS showed that the Ea value of spontaneously dissociated dimer was estimated at 366.2 kJ mol-1 while PLL-g-Dex was about 113.2 kJ mol-1.Dimerization of the stem-loop DNA assisted by PLL-g-Dex was significantly faster than spontaneous dissociation of the dimer.Quick responses were obtained near Tm of the dimer.Self-complementary DNA nanoswitches(DIS25,DIS25-2a,and DIS25-3a series)with high sensitivity driven by a polyelectrolyte were constructed respectively at 60 ?,46 ? and 42 0C,We further designed a double stem-loop DNA(DIS42),and constructed a more complicated nanoswitch system at 37?.Both polyelectrolytes and DNA concentrations are in the nanomole per liter range.The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions.A further application of this sensitive assembly is to construct an aptamer-type drug delivery system,bind or release functional molecules responding to its transconformation.The construction of functional DNA nanomachine driven by cationic comb-type copolymer has important practical significance for the application of DNA nanomachine in biomedical field.